Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: a single center experience

Abstract

The role of panfungal polymerase chain reaction (PCR) assays for diagnosis of invasive fungal disease (IFD) is inadequately defined. We describe the use of an internal transcribed spacer 1 (ITS-1) region-directed panfungal PCR in this context at a tertiary referral transplant center. A retrospective review of patients at Alfred Health, Melbourne, Australia (2009-2014) who had clinical samples referred for panfungal PCR testing was conducted. Baseline patient characteristics, antifungal drug history, fungal culture/histopathology, and radiology results were recorded. For bronchoalveolar lavage (BAL) fluid samples, identification of a fungus other than a Candida spp. was defined as a potential pathogen.Of 138 panfungal PCR tests (108 patients), 41 (30%) were positive for a fungal product. Ninety-seven percent (134/138) of specimens were from immunocompromised hosts. Thirteen percent (19/138) of panfungal PCR positive results were for potential pathogens and potential pathogens were detected more frequently in tissue as compared with BAL (12/13 vs. 6/26; P = .0001). No positive panfungal PCR results were obtained from CSF specimens. If histopathology examination was negative, panfungal PCR identified a potential pathogen in only 12% (11/94) of specimens. For the 20 culture negative/histopathology positive specimens, diagnosis of IFD to causative species level by panfungal PCR occurred in 35% (6/20).Sterile site specimens, in particular tissue, were more frequently panfungal PCR positive for potential pathogens than BAL. The utility of panfungal PCR appears greatest in tissue specimens, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. Based on the results of this study we are now only testing tissue specimens by panfungal PCR.

Diagnosis of invasive fungal diseases in haematology and oncology: 2018 update of the recommendations of the infectious diseases working party of the German society for hematology and medical oncology (AGIHO)

“…For detection of fungi (moulds and yeasts) in tissue samples, in‐house methods and commercial molecular methods have been found to be useful . The utility of PCR appears even greater in tissue specimens than in BAL using an internal transcribed spacer (ITS‐1) region‐directed panfungal PCR, as an adjunct to histopathology to improve diagnostic sensitivity and specificity . Targeted fungal sequencing for culture‐independent diagnosis of IFD was found to have high specificity and sensitivity (both >95%) but the diagnostic yield was dependent on the amount of tissue available for testing (open lung biopsies higher than fine needle aspiration) …”

“…28,114,116,117 The utility of PCR appears even greater in tissue specimens than in BAL using an internal transcribed spacer (ITS-1) region-directed panfungal PCR, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. 118 Targeted fungal sequencing for culture-independent diagnosis of IFD was found to have high specificity and sensitivity (both >95%) but the diagnostic yield was dependent on the amount of tissue…”

Emerging infections caused by non- Aspergillus filamentous fungi

“…Nucleic acid amplification techniques that employ either panfungal PCR assays (combined with DNA sequencing) or fungal species-specific assays may also be used to identify pathogens directly in tissue, blood, serum or sputum [164]. These methods yielded good sensitivity and specificity in small case series [3, 167,168] but methods are not standardized. The application of molecular techniques for the direct detection of Mucorales DNA in fresh and paraffin-embedded tissue specimens is more recentdconventional and real-time PCR 18 and 28S rRNA-targeted assays have been reported with good sensitivity [169e172].…”

Clinical evaluation of an in-house panfungal real-time PCR assay for the detection of fungal pathogens

“…The SepsiTest™ is marketed by Molzym (Molzym Molecular Diagnostic, Bremen, Germany), and targets the 16S rRNA gene for bacteria, and the 18S rRNA gene for fungi. In addition to this assay, few research use only (RUO) tests like the Fungiplex Universal RUO PCR kit (Bruker Daltonik, Bremen, Germany) are commercially available, and a number of in-house panfungal assays have been described [21,[23][24][25][26][27][28][29][30]. We recently published a new panfungal HybProbe real-time PCR assay using the Light-Cycler instrument (Roche), which targets the complete fungal ITS2 region [31].…”